In vivo production of small interfering RNAs that mediate gene silencing

ABSTRACT

The invention provides engineered RNA precursors that when expressed in a cell are processed by the cell to produce targeted small interfering RNAs (siRNAs) that selectively silence targeted genes (by cleaving specific mRNAs) using the cell&#39;s own RNA interference (RNAi) pathway. By introducing nucleic acid molecules that encode these engineered RNA precursors into cells in vivo with appropriate regulatory sequences, expression of the engineered RNA precursors can be selectively controlled both temporally and spatially, i.e., at particular times and/or in particular tissues, organs, or cells.

RELATED APPLICATIONS

This application is a continuation of U.S. Utility application Ser. No.12/727,783, to be issued on Sep. 10, 2013, as U.S. Pat. No. 8,530,438,entitled “In Vivo Production of Small Interfering RNAs that Mediate GeneSilencing” (filed Mar. 19, 2010), which is a continuation of U.S.Utility application Ser. No. 10/195,034, now U.S. Pat. No. 7,691,995,entitled “In Vivo Production of Small Interfering RNAs that Mediate GeneSilencing” (filed Jul. 12, 2002), which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/305,185, entitled “In VivoProduction of Small Interfering RNAs that Mediate Gene Silencing” (filedJul. 12, 2001). The entire contents of the above-referenced patentapplications are incorporated herein by this reference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant no. GM062862awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

This invention relates to ribonucleic acid interference (RNAi), and moreparticularly to RNAi in vivo.

BACKGROUND

RNAi is the sequence-specific, post-transcriptional silencing of agene's expression by double-stranded RNA. RNAi is mediated by 21 to 25nucleotide, double-stranded RNA molecules referred to as smallinterfering RNAs (siRNAs) that are derived by enzymatic cleavage oflong, double-stranded RNA in cells, siRNAs can also be synthesizedchemically or enzymatically outside of cells and then delivered to cells(e.g., by transfection) (see, e.g., Fire et al., 1998, “Potent andspecific genetic interference by double-stranded RNA in Caenorhabditiselegans,” Nature, 391:806-11; Tuschl et al., 1999, “Targeted mRNAdegradation by double-stranded RNA in vitro,” Genes Dev., 13:3191-7;Zamore et al., 2000, “RNAi: double-stranded RNA directs theATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals,” Cell,101:25-33.; Elbashir et al., 2001, “Duplexes of 21-nucleotide RNAsmediate RNA interference in mammalian cell culture,” Nature,411:494-498; and Elbashir et al., 2001, “RNA interference is mediated by21- and 22-nucleotide RNAs,” Genes Dev., 15:188-200.

Double-stranded siRNAs mediate gene silencing by targeting fordisruption or cleavage messenger RNAs (mRNAs) that contain the sequenceof one strand of the siRNA. siRNAs introduced into mammalian cells bytransfection mediate sequence-specific gene silencing, whereas long,double-stranded RNA induces sequence non-specific responses.

SUMMARY

The invention is based on the discovery of new artificial, engineeredRNA precursors, that when expressed in a cell, e.g., in vivo, areprocessed by the cell to produce targeted siRNAs that selectivelysilence target genes (by targeting specific mRNAs for cleavage) usingthe cell's own RNAi pathway. By introducing nucleic acid molecules thatencode these engineered RNA precursors into cells in vivo withappropriate regulatory sequences (e.g., a transgene in a vector such asa plasmid), expression of the engineered RNA precursors can beselectively controlled both temporally and spatially, i.e., atparticular times and/or in particular tissues, organs, or cells.

In general, the invention features an isolated nucleic acid moleculeincluding a regulatory sequence operably linked to a nucleic acidsequence that encodes an engineered ribonucleic acid (RNA) precursor,wherein the precursor includes: (i) a first stem portion comprising asequence of at least 18 nucleotides that is complementary to a sequenceof a messenger RNA (mRNA) of a target gene; (ii) a second stem portioncomprising a sequence of at least 18 nucleotides that is sufficientlycomplementary to the first stem portion to hybridize with the first stemportion to form a duplex stem (e.g., a stem that can be processed by theenzyme Dicer); and (iii) a loop portion that connects the two stemportions. In another aspect, the invention features the engineered RNAitself. The RNA precursor targets a portion of the mRNA of the targetgene, disrupts translation of the mRNA by cleaving the mRNA, and therebyprevents expression of the protein to be inhibited. The target genes canbe, for example, human genes, e.g., mutant human genes, e.g., having apoint mutation, or they can be viral or other genes.

In these molecules and precursors, the first stem portion can be fullycomplementary (i.e., completely complementary) to the mRNA sequence. Inother embodiments, the stem portion can be complementary, i.e., thesequence can be substantially complementary (e.g., there can be no morethan one or two mismatches over a stretch of 20 nucleotides). Similarly,the second stem portion can fully or substantially complementary to thefirst stem portion. The first stem portion can be located at a 5′ or 3′end of the RNA precursor.

In these precursors, the loop portion can include at least 4, 7, or 11,or more nucleotides, and the sequence of the mRNA is located from 100 to300 nucleotides 3′ of the start of translation of the mRNA. The sequenceof the mRNA can be located in a 5′ untranslated region (UTR) or a 3′ UTRof the mRNA. The first and second stem portions can each include about18 to about 30 nucleotides, or about 22 to about 28 nucleotides. Thefirst and second stem portions can each have the same number ofnucleotides, or one of the first and second stem portions can have 1 to4 more nucleotides than the other stem portion. These overhangingnucleotides can all be uracils.

In these nucleic acid molecules, the regulatory sequence can be a PolIII or Pol II promoter, and can be constitutive or inducible. Inspecific embodiments, the engineered RNA precursor can have the sequenceset forth in SEQ ID NO: 1, 2, 3, 4, 5, 8, or 9, and the nucleic acidmolecule can have the sequence set forth in SEQ ID NO: 10, 11, 17, 18,20, or 21, or a complement thereof.

In other embodiments, the invention also features vectors, e.g.,plasmids or viral (e.g., retroviral) vectors, that include the newnucleic acid molecules.

In another aspect, the invention includes host cells, e.g., mammaliancells, that contain the new nucleic acid molecules. The invention alsoincludes transgenes that include the new nucleic acid molecules.

In another aspect of the invention, the invention features transgenic,non-human animals, one or more of whose cells include a transgenecontaining one or more of the new nucleic acid molecules, wherein thetransgene is expressed in one or more cells of the transgenic animalresulting in the animal exhibiting ribonucleic acid interference (RNAi)of the target gene by the engineered RNA precursor. For example, thetransgene can be expressed selectively in one or more cardiac cells,lymphocytes, liver cells, vascular endothelial cells, or spleen cells.In these animals, the regulatory sequence can be constitutive orinducible, or the regulatory sequence can be tissue specific. In someembodiments, the regulatory sequence can a Pol III or Pol II promoter,and can be a an exogenous sequence. These transgenic animals can benon-human primates or rodents, such as mice or rats, or other animals(e.g., other mammals, such as goats or cows; or birds) described herein.

The invention also includes cells derived from the new transgenicanimals. For example, these cells can be a lymphocyte, a hematopoieticcell, a liver cell, a cardiac cell, a vascular endothelial cell, or aspleen cell.

In another aspect, the invention includes methods of inducingribonucleic acid interference (RNAi) of a target gene in a cell, e.g.,in an animal or in culture. The new methods include obtaining atransgenic animal comprising a transgene including a nucleic acidmolecule encoding an engineered RNA precursor and an inducible promoter;and inducing the cell to express the precursor to form a smallinterfering ribonucleic acid (siRNA) within the cell, thereby inducingRNAi of the target gene in the animal.

Alternatively, the methods include obtaining a host cell; culturing thecell; and enabling the cell to express the RNA precursor to form a smallinterfering ribonucleic acid (siRNA) within the cell, thereby inducingRNAi of the target gene in the cell.

A “transgene” is any nucleic acid molecule, which is inserted byartifice into a cell, and becomes part of the genome of the organismthat develops from the cell. Such a transgene may include a gene that ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. The term “transgene” also means a nucleic acid moleculethat includes one or more selected nucleic acid sequences, e.g., DNAs,that encode one or more engineered RNA precursors, to be expressed in atransgenic organism, e.g., animal, which is partly or entirelyheterologous, i.e., foreign, to the transgenic animal, or homologous toan endogenous gene of the transgenic animal, but which is designed to beinserted into the animal's genome at a location which differs from thatof the natural gene. A transgene includes one or more promoters and anyother DNA, such as introns, necessary for expression of the selectednucleic acid sequence, all operably linked to the selected sequence, andmay include an enhancer sequence.

A “transformed cell” is a cell into which (or into an ancestor of which)has been introduced, by means of recombinant DNA techniques, a nucleicacid molecule or transgene encoding an engineered RNA precursor.

As used herein, the term “operably linked” means that a selected nucleicacid sequence, e.g., encoding an engineered RNA precursor, is inproximity with a promoter, e.g., a tissue-specific promoter, to allowthe promoter to regulate expression of the selected nucleic acidsequence. In addition, the promoter is located upstream of the selectednucleic acid sequence in terms of the direction of transcription andtranslation.

By “promoter” is meant a nucleic acid sequence that is sufficient todirect transcription. A tissue-specific promoter affects expression ofthe selected nucleic acid sequence in specific cells, e.g.,hematopoietic cells, or cells of a specific tissue within an animal,e.g., cardiac, muscle, or vascular endothelium. The term also coversso-called “leaky” promoters, which regulate expression of a selectednucleic acid sequence primarily in one tissue, but cause expression inother tissues as well. Such promoters also may include additional DNAsequences that are necessary for expression, such as introns andenhancer sequences.

By “transgenic” is meant any cell that includes a nucleic acid, e.g.,DNA sequence, that is inserted by artifice into a cell and becomes partof the genome of an organism that develops from that cell. A “transgenicanimal” means an animal that includes a transgene that is inserted intoan embryonal cell and becomes a part of the genome of the animal whichdevelops from that cell, or an offspring of such an animal. In thetransgenic animals described herein, the transgene causes specifictissue cells to express an engineered RNA precursor. Any animal that canbe produced by transgenic technology is included in the invention,although mammals are preferred. Preferred mammals include non-humanprimates, sheep, goats, horses, cattle, pigs, rabbits, and rodents suchas guinea pigs, hamsters, rats, gerbils, and, preferably, mice.

An “isolated nucleic acid molecule or sequence” is a nucleic acidmolecule or sequence that is not immediately contiguous with both of thecoding sequences with which it is immediately contiguous (one on the 5′end and one on the 3′ end) in the naturally occurring genome of theorganism from which it is derived. The term therefore includes, forexample, a recombinant DNA or RNA that is incorporated into a vector;into an autonomously replicating plasmid or virus; or into the genomicDNA of a prokaryote or eukaryote, or which exists as a separate molecule(e.g., a cDNA or a genomic DNA fragment produced by PCR or restrictionendonuclease treatment) independent of other sequences. It also includesa recombinant DNA that is part of a hybrid gene encoding an additionalpolypeptide sequence.

A “target gene” is a gene whose expression is to be selectivelyinhibited or “silenced.” This silencing is achieved by cleaving the mRNAof the target gene by an siRNA that is created from an engineered RNAprecursor by a cell's RNAi system. One portion or segment of a duplexstem of the RNA precursor is an anti-sense strand that is complementary,e.g., fully complementary, to a section of about 18 to about 40 or morenucleotides of the mRNA of the target gene.

The term “engineered,” as in an engineered RNA precursor, or anengineered nucleic acid molecule, indicates that the precursor ormolecule is not found in nature, in that all or a portion of the nucleicacid sequence of the precursor or molecule is created or selected byman. Once created or selected, the sequence can be replicated,translated, transcribed, or otherwise processed by mechanisms within acell. Thus, an RNA precursor produced within a cell from a transgenethat includes an engineered nucleic acid molecule is an engineered RNAprecursor.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The invention provides several advantages. For example, the inventionimproves on and overcomes a significant deficiency in the prior art.Prior methods for inducing RNAi in mammalian cells using siRNAs wererestricted to cell cultures. The new methods extend RNAi to wholeanimals, e.g., mammals, and thus allow RNAi to be targeted to specificcell types, organs, or tissues, and/or to specific developmental stages.

In addition, this technology simplifies and lowers the cost of siRNAconstruction, because DNA molecules are relatively inexpensive to make.Thus, large populations of plasmids or other vectors can be prepared,each containing a nucleic acid molecule that encodes an engineered RNAprecursor that targets a particular gene, can be easily prepared, e.g.,in an array format. In addition, the new nucleic acid molecules can beintroduced into a variety of cells, which can be cultured in vitro usingknown techniques. Furthermore, the new methods enable the long-term,e.g., permanent, reduction of targeted gene expression in cell lines,because siRNAs are transient, but a transgenic hairpin provides along-lasting supply of siRNAs.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the dual nature of the stRNA and siRNApathways.

FIG. 2A is a schematic representation of a wild-type, stRNA precursor(SEQ ID NO: 1).

FIGS. 2B to 2E are schematic representations of synthetic, engineeredRNA precursors (SEQ ID NOS:2, 3, 4, and 5).

FIG. 3 is an autoradiograph showing the results of an assay fordetermining whether an engineered RNA precursor can promote cleavage ofthe target mRNA in vitro in a standard RNAi reaction.

FIGS. 4A to 4C are schematic representations of synthetic luciferasesiRNA (4A; SEQ ID NOS: 6 and 7), and 5′ and 3′ synthetic, engineered RNAprecursors (4B; SEQ ID NO:8; and 4C; SEQ ID NO:9).

FIG. 4D is a schematic representation of a chimeric target mRNA for anin vitro luciferase/let-7 RNAi reaction. The sites of siRNA-directedtarget cleavage are indicated by “scissors.”

FIG. 4E is an autoradiograph showing the results of an assay fordetermining whether the 5′ and 3′ synthetic, engineered RNA precursorsof FIGS. 4B and 4C can promote cleavage of the target mRNA in vitro in astandard RNAi reaction.

FIG. 5 is a schematic diagram of transgene encoding an engineered RNAprecursor (SEQ ID NO:2) and the transcription and processing of theprecursor to form a double-stranded siRNA (SEQ ID NO:7 and SEQ IDNO:12).

DETAILED DESCRIPTION

Small temporal RNAs (stRNAs), also known as microRNAs (miRNAs), such aslin-4 and let-7 in Caenorhabditis elegans and let-7 in Drosophilamelanogaster and humans encode no protein, but instead appear to blockthe productive translation of mRNA by binding sequences in the 3′untranslated region (3′ UTR) of their target mRNAs. As described inHutvágner et al., Science, 293:834 (Jul. 12, 2001), let-7 RNA inDrosophila has been shown to be cleaved from a larger precursortranscript, which is similar to the generation of small RNAs from alonger, structured precursor double-stranded RNA in the RNA interference(RNAi) pathway.

Like siRNA, stRNAs are also 21-25 nucleotides long, but unlike siRNAs,are single-stranded and do not mediate gene silencing via target mRNAcleavage. As shown in FIG. 1, the RNAi and stRNA pathways intersect;both require the RNA processing enzyme Dicer to produce the active smallRNA components that repress gene expression. Dicer and perhaps otherproteins act on pre-stRNAs to yield mature, single-stranded stRNAs thatrepress mRNA translation. In RNAi, Dicer cleaves long, double-strandedRNA to yield siRNA duplexes that mediate targeted mRNA destruction.

Whereas long, double-stranded RNAs are cleaved symmetrically by Dicer togenerate duplex siRNAs, current evidence suggests that stRNAs arecleaved asymmetrically to generate only a single-stranded stRNA. stRNAprecursors are stem-loop RNAs that do not mediate target cleavage orprovoke the sequence non-specific responses induced by long,double-stranded RNA. On the other hand, the invention provides new,engineered RNA precursors that when processed within a cell generatesiRNAs that mediated target cleavage. These siRNAs can be double- orsingle-stranded, as long as they mediate cleavage of the target mRNA.Such engineered RNA precursors can be expressed in transgenic mammals ina cell-type-specific or developmental-stage-specific manner to induceRNAi in a specific cell or cells at a defined time.

A Drosophila embryo lysate that mediates RNAi in vitro (Tuschl et al.,(1999) cited supra), which process double-stranded RNA into siRNA(Zamore et al., (2000) cited supra), and pre-let-7-stRNA into maturelet-7 stRNA (Hutvágner et al., (2001, cited supra), can be used to assaythe ability of an engineered RNA precursor to mediate RNAi in vitro.This assay allows testing of the new engineered RNA precursors. The newengineered precursors differ from naturally occurring, wild-type stRNAprecursors by various modifications and by the fact that one portion oftheir duplex stem comprises a nucleic acid sequence that iscomplementary, preferably fully complementary, to a portion of the mRNAof a target gene.

Engineered RNA Precursors that Generate siRNAs

Naturally-occurring stRNA precursors (pre-stRNA) have certain elementsor components that are illustrated in FIG. 2A, which shows an stRNAprecursor for let-7 (pre-let-7). Each precursor is a single strand thatforms a duplex stem including two portions that are generallycomplementary, and a loop, that connects the two portions of the stem.In typical pre-stRNAs, the stem includes one or more bulges, e.g., extranucleotides that create a single nucleotide “loop” in one portion of thestem, and/or one or more unpaired nucleotides that create a gap in thehybridization of the two portions of the stem to each other.

Engineered RNA precursors of the invention are artificial constructsthat are similar to naturally occurring pre-stRNAs, but differ from thewild-type precursor sequences in a number of ways. The key difference isthat one portion of the duplex stem is a nucleic acid sequence that iscomplementary (or anti-sense) to the target mRNA. Thus, engineered RNAprecursors include a duplex stem with two portions and a loop connectingthe two stem portions. The two stem portions are about 18 or 19 to about25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. When usedin mammalian cells, the length of the stem portions should be less thanabout 30 nucleotides to avoid provoking non-specific responses like theinterferon pathway. In non-mammalian cells, the stem can be longer than30 nucleotides. In fact, the stem can include much larger sectionscomplementary to the target mRNA (up to, and including the entire mRNA).The two portions of the duplex stem must be sufficiently complementaryto hybridize to form the duplex stem. Thus, the two portions can be, butneed not be, fully or perfectly complementary. In addition, the two stemportions can be the same length, or one portion can include an overhangof 1, 2, 3, or 4 nucleotides. The overhanging nucleotides can include,for example, uracils (Us), e.g., all Us.

Other differences from natural pre-stRNA sequences include, but are notlimited to, deleting unpaired or bulged nucleotides, introducingadditional base-paired nucleotides to one or both of the stem portions,modifying the loop sequence to increase or decrease the number of pairednucleotides, or replacing all or part of the loop sequence with atetraloop or other loop sequences. Thus, the loop in the engineered RNAprecursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, ormore nucleotides in length. Tetraloop sequences can include, but are notlimited to, the sequences GNRA (SEQ ID NO: 13), where N is anynucleotide and R is a purine nucleotide, GGGG (SEQ ID NO: 14), and UUUU(SEQ ID NO:15).

Four examples of such engineered RNA precursors are illustrated in FIGS.2B to 2E. FIGS. 2B and 2C illustrate engineered precursors in which thestem portions have had all unpaired and bulging nucleotides removed orpaired, but the loop is the same as the wild-type loop in the pre-stRNA.FIGS. 2D and 2E illustrate two engineered RNA precursors with atetraloop. In FIG. 2D, the tetraloop UUUU (SEQ ID NO: 15) replaces aportion of the wild-type loop in FIG. 2A. In FIG. 2E, the tetraloop GGGG(SEQ ID NO: 14) replaces the entire wild-type loop sequence.

FIGS. 4B and 4C illustrate additional engineered RNA precursors. Eachengineered RNA precursor includes in its stem a sequence that isperfectly complementary to a portion of the sequence of the fireflyluciferase mRNA. In FIG. 4B (SEQ ID NO:8), this region is shown in boldtype, and is located on the 3′ side of the stem. In FIG. 4C (SEQ IDNO:9), this complementary sequence is on the 5′ side of the stem. Unlikethe naturally-occurring pre-let-7 RNA, these engineered RNA precursorshave fully complementary stems, and direct RNAi against the lucifersemRNA.

In addition, modification of the naturally occurring stRNA precursor togenerate an engineered RNA precursor (pre-siRNA) includes altering thesequence of the RNA to include the sequences of the desired siRNAduplex. The desired siRNA duplex, and thus both of the two stem portionsin the engineered RNA precursor, are selected by methods known in theart. These include, but are not limited to, selecting an 18, 19, 20, 21nucleotide, or longer, sequence from the target gene mRNA sequence froma region 100 to 200 or 300 nucleotides on the 3′ side of the start oftranslation. In general, the sequence can be selected from any portionof the mRNA from the target gene, such as the 5′ UTR (untranslatedregion), coding sequence, or 3′ UTR. This sequence can optionally followimmediately after a region of the target gene containing two adjacent AAnucleotides. The last two nucleotides of the 21 or so nucleotidesequence can be selected to be UU (so that the anti-sense strand of thesiRNA begins with UU). This 21 or so nucleotide sequence is used tocreate one portion of a duplex stem in the engineered RNA precursor.This sequence can replace a stem portion of a wild-type pre-stRNAsequence, e.g., enzymatically, or is included in a complete sequencethat is synthesized. For example, one can synthesize DNAoligonucleotides that encode the entire stem-loop engineered RNAprecursor, or that encode just the portion to be inserted into theduplex stem of the precursor, and using restriction enzymes to build theengineered RNA precursor construct, e.g., from a wild-type pre-stRNA.

Engineered RNA precursors include in the duplex stem the 21-22 or sonucleotide sequences of the siRNA desired to be produced in vivo. Thus,the stem portion of the engineered RNA precursor includes at least 18 or19 nucleotide pairs corresponding to the sequence of an exonic portionof the gene whose expression is to be reduced or inhibited. The two 3′nucleotides flanking this region of the stem are chosen so as tomaximize the production of the siRNA from the engineered RNA precursor,and to maximize the efficacy of the resulting siRNA in targeting thecorresponding mRNA for destruction by RNAi in vivo and in vitro.

Another defining feature of these engineered RNA precursors is that as aconsequence of their length, sequence, and/or structure, they do notinduce sequence non-specific responses, such as induction of theinterferon response or apoptosis, or that they induce a lower level ofsuch sequence non-specific responses than long, double-stranded RNA(>150 bp) currently used to induce RNAi. For example, the interferonresponse is triggered by dsRNA longer than 30 base pairs.

Transgenes Encoding Engineered RNA Precursors

The new engineered RNA precursors can be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.).These synthetic, engineered RNA precursors can be used directly asdescribed below or cloned into expression vectors by methods known inthe field.

The engineered RNA precursors should be delivered to cells in vitro orin vivo in which it is desired to target a specific mRNA fordestruction. A number of methods have been developed for delivering DNAor RNA to cells. For example, for in vivo delivery, molecules can beinjected directly into a tissue site or administered systemically. Invitro delivery includes methods known in the art such as electroporationand lipofection.

To achieve intracellular concentrations of the nucleic acid moleculesufficient to suppress expression of endogenous mRNAs, one can use, forexample, a recombinant DNA construct in which the oligonucleotide isplaced under the control of a strong Pol III (e.g., U6 or PolIII H1-RNApromoter) or Pol II promoter. The use of such a construct to transfecttarget cells in vitro or in vivo will result in the transcription ofsufficient amounts of the engineered RNA precursor to lead to theproduction of an siRNA that can target a corresponding mRNA sequence forcleavage by RNAi to decrease the expression of the gene encoding thatmRNA. For example, a vector can be introduced in vivo such that it istaken up by a cell and directs the transcription of an engineered RNAprecursor. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredstRNA precursor.

Such vectors can be constructed by recombinant DNA technology methodsknown in the art. Vectors can be plasmid, viral, or other vectors knownin the art such as those described herein, used for replication andexpression in mammalian cells or other targeted cell types. The nucleicacid sequences encoding the engineered RNA precursors can be preparedusing known techniques. For example, two synthetic DNA oligonucleotidescan be synthesized to create a novel gene encoding the entire engineeredRNA precursor. The DNA oligonucleotides, which will pair, leavingappropriate ‘sticky ends’ for cloning, can be inserted into arestriction site in a plasmid that contains a promoter sequence (e.g., aPol II or a Pol III promoter) and appropriate terminator sequences 3′ tothe engineered RNA precursor sequences (e.g., a cleavage andpolyadenylation signal sequence from SV40 or a Pol III terminatorsequence).

The invention also encompasses genetically engineered host cells thatcontain any of the foregoing expression vectors and thereby express thenucleic acid molecules of the invention in the host cell. The host cellscan be cultured using known techniques and methods (see, e.g., Cultureof Animal Cells (R. I. Freshney, Alan R. Liss, Inc. 1987); MolecularCloning, Sambrook et al. (Cold Spring Harbor Laboratory Press, 1989)).

Successful introduction of the vectors of the invention into host cellscan be monitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection canbe indicated using markers that provider the transfected cell withresistance to specific environmental factors (e.g., antibiotics anddrugs), such as hygromycin B resistance, e.g., in insect cells and inmammalian cells.

Regulatory Sequences

The expression of the engineered RNA precursors is driven by regulatorysequences, and the vectors of the invention can include any regulatorysequences known in the art to act in mammalian cells, e.g., murinecells; in insect cells; in plant cells; or other cells. The termregulatory sequence includes promoters, enhancers, and other expressioncontrol elements. It will be appreciated that the appropriate regulatorysequence depends on such factors as the future use of the cell ortransgenic animal into which a sequence encoding an engineered RNAprecursor is being introduced, and the level of expression of thedesired RNA precursor. A person skilled in the art would be able tochoose the appropriate regulatory sequence. For example, the transgenicanimals described herein can be used to determine the role of a testpolypeptide or the engineered RNA precursors in a particular cell type,e.g., a hematopoietic cell. In this case, a regulatory sequence thatdrives expression of the transgene ubiquitously, or ahematopoietic-specific regulatory sequence that expresses the transgeneonly in hematopoietic cells, can be used. Expression of the engineeredRNA precursors in a hematopoietic cell means that the cell is nowsusceptible to specific, targeted RNAi of a particular gene. Examples ofvarious regulatory sequences are described below.

The regulatory sequences can be inducible or constitutive. Suitableconstitutive regulatory sequences include the regulatory sequence of ahousekeeping gene such as the α-actin regulatory sequence, or may be ofviral origin such as regulatory sequences derived from mouse mammarytumor virus (MMTV) or cytomegalovirus (CMV).

Alternatively, the regulatory sequence can direct transgene expressionin specific organs or cell types (see, e.g., Lasko et al., 1992, Proc.Natl. Acad. Sci. USA 89:6232). Several tissue-specific regulatorysequences are known in the art including the albumin regulatory sequencefor liver (Pinkert et al., 1987, Genes Dev. 1:268-276); the endothelinregulatory sequence for endothelial cells (Lee, 1990, J. Biol. Chem.265:10446-50); the keratin regulatory sequence for epidermis; the myosinlight chain-2 regulatory sequence for heart (Lee et al., 1992, J. Biol.Chem. 267:15875-85), and the insulin regulatory sequence for pancreas(Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515), or thevav regulatory sequence for hematopoietic cells (Oligvy et al., 1999,Proc. Natl. Acad. Sci. USA 96:14943-14948). Another suitable regulatorysequence, which directs constitutive expression of transgenes in cellsof hematopoietic origin, is the murine MHC class I regulatory sequence(Morello et al., 1986, EMBO J. 5:1877-1882). Since MHC expression isinduced by cytokines, expression of a test gene operably linked to thisregulatory sequence can be upregulated in the presence of cytokines.

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals such as mice, include regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D-1-thiogalactopyranoside (IPTG) (collectively referredto as “the regulatory molecule”). Each of these expression systems iswell described in the literature and permits expression of the transgenethroughout the animal in a manner controlled by the presence or absenceof the regulatory molecule. For a review of inducible expressionsystems, see, e.g., Mills, 2001, Genes Devel. 15:1461-1467, andreferences cited therein.

The regulatory elements referred to above include, but are not limitedto, the cytomegalovirus hCMV immediate early gene, the early or latepromoters of SV40 adenovirus (Bernoist et al., Nature, 290:304, 1981),the tet system, the lac system, the tr system, the TAC system, the TRCsystem, the major operator and promoter regions of phage A, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeastα-mating factors. Additional promoters include the promoter contained inthe 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell22:787-797, 1988); the herpes thymidine kinase promoter (Wagner et al.,Proc. Natl. Acad. Sci. USA 78:1441, 1981); or the regulatory sequencesof the metallothionein gene (Brinster et al., Nature 296:39, 1988).

Assay for Testing Engineered RNA Precursors

Drosophila embryo lysates can be used to determine if an engineered RNAprecursor was, in fact, the direct precursor of a mature stRNA or siRNA.This lysate assay is described in Tuschl et al., 1999, supra, Zamore etal., 2000, supra, and Hutvágner et al. 2001, supra. These lysatesrecapitulate RNAi in vitro, thus permitting investigation into whetherthe proposed precursor RNA was cleaved into a mature stRNA or siRNA byan RNAi-like mechanism. Briefly, the precursor RNA is incubated withDrosophila embryo lysate for various times, then assayed for theproduction of the mature siRNA or stRNA by primer extension or Northernhybridization. As in the in vivo setting, mature RNA accumulates in thecell-free reaction. Thus, an RNA corresponding to the proposed precursorcan be shown to be converted into a mature stRNA or siRNA duplex in theDrosophila embryo lysate.

Furthermore, an engineered RNA precursor can be functionally tested inthe Drosophila embryo lysates. In this case, the engineered RNAprecursor is incubated in the lysate in the presence of a 5′radiolabeled target mRNA in a standard in vitro RNAi reaction forvarious lengths of time. The target mRNA can be 5 radiolabeled usingguanylyl transferase (as described in Tuschl et al., 1999, supra andreferences therein) or other suitable methods. The products of the invitro reaction are then isolated and analyzed on a denaturing acrylamideor agarose gel to determine if the target mRNA has been cleaved inresponse to the presence of the engineered RNA precursor in thereaction. The extent and position of such cleavage of the mRNA targetwill indicate if the engineering of the precursor created a pre-siRNAcapable of mediating sequence-specific RNAi.

Transgenic Animals

Engineered RNA precursors of the invention can be expressed intransgenic animals. These animals represent a model system for the studyof disorders that are caused by, or exacerbated by, overexpression orunderexpression (as compared to wild-type or normal) of nucleic acids(and their encoded polypeptides) targeted for destruction by theengineered RNA precursor products (siRNAs), and for the development oftherapeutic agents that modulate the expression or activity of nucleicacids or polypeptides targeted for destruction.

Transgenic animals can be farm animals (pigs, goats, sheep, cows,horses, rabbits, and the like), rodents (such as rats, guinea pigs, andmice), non-human primates (for example, baboons, monkeys, andchimpanzees), and domestic animals (for example, dogs and cats).Invertebrates such as Caenorhabditis elegans or Drosophila can be usedas well as non-mammalian vertebrates such as fish (e.g., zebrafish) orbirds (e.g., chickens). Engineered RNA precursors with stems of 18 to 30nucleotides in length are preferred for use in mammals, such as mice.

A transgenic founder animal can be identified based upon the presence ofa transgene that encodes the new RNA precursors in its genome, and/orexpression of the transgene in tissues or cells of the animals, forexample, using PCR or Northern analysis. Expression is confirmed by adecrease in the expression (RNA or protein) of the target sequence.

A transgenic founder animal can be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding the RNA precursors can further be bred to othertransgenic animals carrying other transgenes. In addition, cellsobtained from the transgenic founder animal or its offspring can becultured to establish primary, secondary, or immortal cell linescontaining the transgene.

Procedures for Making Transgenic, Non-Human Animals

A number of methods have been used to obtain transgenic, non-humananimals, which are animals that have gained an additional gene by theintroduction of a transgene into their cells (e.g., both the somatic andgerm cells), or into an ancestor's germ line. In some cases, transgenicanimals can be generated by commercial facilities (e.g., The TransgenicDrosophila Facility at Michigan State University, The TransgenicZebrafish Core Facility at the Medical College of Georgia (Augusta,Ga.), and Xenogen Biosciences (St. Louis, Mo.). In general, theconstruct containing the transgene is supplied to the facility forgenerating a transgenic animal.

Methods for generating transgenic animals include introducing thetransgene into the germ line of the animal. One method is bymicroinjection of a gene construct into the pronucleus of an early stageembryo (e.g., before the four-cell stage; Wagner et al., 1981, Proc.Natl. Acad. Sci. USA 78:5016; Brinster et al., 1985, Proc. Natl. Acad.Sci. USA 82:4438). Alternatively, the transgene can be introduced intothe pronucleus by retroviral infection. A detailed procedure forproducing such transgenic mice has been described (see e.g., Hogan etal., Manipulating the Mouse Embryo, Cold Spring Harbour Laboratory, ColdSpring Harbour, N.Y. (1986); U.S. Pat. No. 5,175,383 (1992)). Thisprocedure has also been adapted for other animal species (e.g., Hammeret al., 1985, Nature 315:680; Murray et al., 1989, Reprod. Fert. Devl.1:147; Pursel et al., 1987, Vet. Imumunol. Histopath. 17:303; Rexroad etal., 1990, J. Reprod. Fert. 41 (suppl):119; Rexroad et al., 1989, Molec.Reprod. Devl. 1:164; Simons et al., 1988, BioTechnology 6:179; Vize etal., 1988, J. Cell. Sci. 90:295; and Wagner, 1989, J. Cell. Biochem. 13B(suppl):164).

In brief, the procedure involves introducing the transgene into ananimal by microinjecting the construct into the pronuclei of thefertilized mammalian egg(s) to cause one or more copies of the transgeneto be retained in the cells of the developing mammal(s). Followingintroduction of the transgene construct into the fertilized egg, the eggmay be incubated in vitro for varying amounts of time, or reimplanted ain surrogate host, or both. One common method is to incubate the embryosin vitro for about 1-7 days, depending on the species, and thenreimplant them into the surrogate host. The presence of the transgene inthe progeny of the transgenically manipulated embryos can be tested bySouthern blot analysis of a segment of tissue.

Another method for producing germ-line transgenic animals is through theuse of embryonic stem (ES) cells. The gene construct can be introducedinto embryonic stem cells by homologous recombination (Thomas et al.,1987, Cell 51:503; Capecchi, Science 1989, 244:1288; Joyner et al.,1989, Nature 338:153) in a transcriptionally active region of thegenome. A suitable construct can also be introduced into embryonic stemcells by DNA-mediated transfection, such as by electroporation (Ausubelet al., Current Protocols in Molecular Biology, John Wiley & Sons,1987). Detailed procedures for culturing embryonic stem cells (e.g.,ES-D3, ATCC#CCL-1934, ES-E14TG2a, ATCC#CCL-1821, American Type CultureCollection, Rockville, Md.) and methods of making transgenic animalsfrom embryonic stem cells can be found in Teratocarcinomas and EmbryonicStem Cells, A Practical Approach, ed. E. J. Robertson (IRL Press, 1987).In brief, the ES cells are obtained from pre-implantation embryoscultured in vitro (Evans et al., 1981, Nature 292:154-156). Transgenescan be efficiently introduced into ES cells by DNA transfection or byretrovirus-mediated transduction. The resulting transformed ES cells canthereafter be combined with blastocysts from a non-human animal. The EScells colonize the embryo and contribute to the germ line of theresulting chimeric animal.

In the above methods, the transgene can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can beincorporated and inherited as a transgene integrated into the hostgenome. The transgene can also be constructed to permit it to beinherited as an extrachromosomal plasmid (Gassmann et al., 1995, Proc.Natl. Acad. Sci. USA 92:1292). A plasmid is a DNA molecule that canreplicate autonomously in a host.

The transgenic, non-human animals can also be obtained by infecting ortransfecting cells either in vivo (e.g., direct injection), ex vivo(e.g., infecting the cells outside the host and later reimplanting), orin vitro (e.g., infecting the cells outside host), for example, with arecombinant viral vector carrying a gene encoding the engineered RNAprecursors. Examples of suitable viral vectors include recombinantretroviral vectors (Valerio et al., 1989, Gene 84:419; Scharfman et al.,1991, Proc. Natl. Acad. Sci. USA 88:462; Miller and Buttimore, 1986,Mol. Cell. Biol. 6:2895), recombinant adenoviral vectors (Freidman etal., 1986, Mol. Cell. Biol. 6:3791; Levrero et al., 1991, Gene 101:195),and recombinant Herpes simplex viral vectors (Fink et al., 1992, HumanGene Therapy 3:11). Such methods are also useful for introducingconstructs into cells for uses other than generation of transgenicanimals.

Other approaches include insertion of transgenes encoding the newengineered RNA precursors into viral vectors including recombinantadenovirus, adeno-associated virus, and herpes simplex virus-1, orrecombinant bacterial or eukaryotic plasmids. Viral vectors transfectcells directly. Other approaches include delivering the transgenes, inthe form of plasmid DNA, with the help of, for example, cationicliposomes (lipofectin) or derivatized (e.g. antibody conjugated)polylysine conjugates, gramacidin S, artificial viral envelopes, orother such intracellular carriers, as well as direct injection of thetransgene construct or CaPO₄ precipitation carried out in vivo. Suchmethods can also be used in vitro to introduce constructs into cells foruses other than generation of transgenic animals.

Retrovirus vectors and adeno-associated virus vectors can be used as arecombinant gene delivery system for the transfer of exogenous genes invivo or in vitro. These vectors provide efficient delivery of genes intocells, and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host. The development of specialized cell lines(termed “packaging cells”) which produce only replication-defectiveretroviruses has increased the utility of retroviruses for gene therapy,and defective retroviruses are characterized for use in gene transferfor gene therapy purposes (for a review see Miller, 1990, Blood 76:271).A replication-defective retrovirus can be packaged into virions whichcan be used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals.

Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM whichare known to those skilled in the art. Examples of suitable packagingvirus lines for preparing both ecotropic and amphotropic retroviralsystems include Psi-Crip, Psi-Cre, Psi-2 and Psi-Am. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including epithelial cells, in vitro and/or in vivo (see forexample Eglitis, et al., 1985, Science 230:1395-1398; Danos andMulligan, 1988, Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al.,1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990,Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al., 1991, Proc. Natl.Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci.USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

In another example, recombinant retroviral vectors capable oftransducing and expressing genes inserted into the genome of a cell canbe produced by transfecting the recombinant retroviral genome intosuitable packaging cell lines such as PA317 and Psi-CRIP (Cornette etal., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl.Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used toinfect a wide variety of cells and tissues in susceptible hosts (e.g.,rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. InfectiousDisease, 166:769), and also have the advantage of not requiringmitotically active cells for infection.

Another viral gene delivery system useful in the present invention alsoutilizes adenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See, for example, Berkner et al. (1988,BioTechniques 6:616), Rosenfeld et al. (1991, Science 252:431-434), andRosenfeld et al. (1992, Cell 68:143-155). Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in theart. Recombinant adenoviruses can be advantageous in certaincircumstances in that they are not capable of infecting nondividingcells and can be used to infect a wide variety of cell types, includingepithelial cells (Rosenfeld et al., 1992, cited supra). Furthermore, thevirus particle is relatively stable and amenable to purification andconcentration, and as above, can be modified to affect the spectrum ofinfectivity. Additionally, introduced adenoviral DNA (and foreign DNAcontained therein) is not integrated into the genome of a host cell butremains episomal, thereby avoiding potential problems that can occur asa result of insertional mutagenesis in situ where introduced DNA becomesintegrated into the host genome (e.g., retroviral DNA). Moreover, thecarrying capacity of the adenoviral genome for foreign DNA is large (upto 8 kilobases) relative to other gene delivery vectors (Berkner et al.cited supra; Haj-Ahmand and Graham, 1986, J. Virol. 57:267).

Yet another viral vector system useful for delivery of the subjecttransgenes is the adeno-associated virus (AAV). Adeno-associated virusis a naturally occurring defective virus that requires another virus,such as an adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. For a review, see Muzyczka etal. (1992, Curr. Topics in Micro. and Immunol. 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992, Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al., 1989, J. Virol. 63:3822-3828; and McLaughlin et al.(1989, J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of anengineered RNA precursor of the invention in the tissue of an animal.Most non-viral methods of gene transfer rely on normal mechanisms usedby mammalian cells for the uptake and intracellular transport ofmacromolecules. In preferred embodiments, non-viral gene deliverysystems of the present invention rely on endocytic pathways for theuptake of the subject gene of the invention by the targeted cell.Exemplary gene delivery systems of this type include liposomal derivedsystems, poly-lysine conjugates, and artificial viral envelopes. Otherembodiments include plasmid injection systems such as are described inMeuli et al., (2001) J. Invest. Dermatol., 116(1):131-135; Cohen et al.,(2000) Gene Ther., 7(22):1896-905; and Tam et al., (2000) Gene Ther.,7(21):1867-74.

In a representative embodiment, a gene encoding an engineered RNAprecursor of the invention can be entrapped in liposomes bearingpositive charges on their surface (e.g., lipofectins) and (optionally)which are tagged with antibodies against cell surface antigens of thetarget tissue (Mizuno et al., (1992) No Shinkei Geka, 20:547-551; PCTpublication WO91/06309; Japanese patent application 1047381; andEuropean patent publication EP-A-43075).

Animals harboring the transgene can be identified by detecting thepresence of the transgene in genomic DNA (e.g., using Southernanalysis). In addition, expression of the engineered RNA precursor canbe detected directly (e.g., by Northern analysis). Expression of thetransgene can also be confirmed by detecting a decrease in the amount ofprotein corresponding to the targeted sequence. When the transgene isunder the control of an inducible or developmentally regulated promoter,expression of the target protein is decreased when the transgene isinduced or at the developmental stage when the transgene is expressed,respectively.

Clones of Transgenic Animals

Clones of the non-human transgenic animals described herein can beproduced according to the methods described in Wilmut et al. ((1997)Nature, 385:810-813) and PCT publication Nos. WO 97/07668 and WO97/07669. In brief, a cell, e.g., a somatic cell from the transgenicanimal, can be isolated and induced to exit the growth cycle and enterthe G_(o) phase to become quiescent. The quiescent cell can then befused, e.g., through the use of electrical pulses, to an enucleatedoocyte from an animal of the same species from which the quiescent cellis isolated. The reconstructed oocyte is then cultured such that itdevelops into a morula or blastocyte and is then transferred to apseudopregnant female foster animal. Offspring borne of this femalefoster animal will be clones of the animal from which the cell, e.g.,the somatic cell, was isolated.

Once the transgenic animal is produced, cells of the transgenic animaland cells from a control animal are screened to determine the presenceof an RNA precursor nucleic acid sequence, e.g., using polymerase chainreaction (PCR). Alternatively, the cells can be screened to determine ifthe RNA precursor is expressed (e.g., by standard procedures such asNorthern blot analysis or reverse transcriptase-polymerase chainreaction (RT-PCR); Sambrook et al., Molecular Cloning—A LaboratoryManual, (Cold Spring Harbor Laboratory, 1989)).

The transgenic animals of the present invention can be homozygous orheterozygous, and one of the benefits of the invention is that thetarget mRNA is effectively degraded even in heterozygotes. The presentinvention provides for transgenic animals that carry a transgene of theinvention in all their cells, as well as animals that carry a transgenein some, but not all of their cells. That is, the invention provides formosaic animals. The transgene can be integrated as a single transgene orin concatamers, e.g., head-to-head tandems or head-to-tail tandems.

For a review of techniques that can be used to generate and assesstransgenic animals, skilled artisans can consult Gordon (Intl. Rev.Cytol. 115:171-229, 1989), and may obtain additional guidance from, forexample: Hogan et al. “Manipulating the Mouse Embryo” (Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1986; Krimpenfort et al.,Bio/Technology 9:86, 1991; Palmiter et al., Cell 41:343, 1985; Kraemeret al., “Genetic Manipulation of the Early Mammalian Embryo,” ColdSpring Harbor Press, Cold Spring Harbor, N.Y., 1985; Hammer et al.,Nature 315:680, 1985; Purcel et al., Science, 244:1281, 1986; Wagner etal., U.S. Pat. No. 5,175,385; and Krimpenfort et al., U.S. Pat. No.5,175,384.

Transgenic Plants

Among the eukaryotic organisms featured in the invention are plantscontaining an exogenous nucleic acid that encodes an engineered RNAprecursor of the invention.

Accordingly, a method according to the invention comprises making aplant having a nucleic acid molecule or construct, e.g., a transgene,described herein. Techniques for introducing exogenous nucleic acidsinto monocotyledonous and dicotyledonous plants are known in the art,and include, without limitation, Agrobacterium-mediated transformation,viral vector-mediated transformation, electroporation and particle guntransformation, see, e.g., U.S. Pat. Nos. 5,204,253 and 6,013,863. If acell or tissue culture is used as the recipient tissue fortransformation, plants can be regenerated from transformed cultures bytechniques known to those skilled in the art. Transgenic plants can beentered into a breeding program, e.g., to introduce a nucleic acidencoding a polypeptide into other lines, to transfer the nucleic acid toother species or for further selection of other desirable traits.Alternatively, transgenic plants can be propagated vegetatively forthose species amenable to such techniques. Progeny includes descendantsof a particular plant or plant line. Progeny of a plant include seedsformed on F₁, F₂, F₃, and subsequent generation plants, or seeds formedon BC₁, BC₂, BC₃, and subsequent generation plants. Seeds produced by atransgenic plant can be grown and then selfed (or outcrossed and selfed)to obtain seeds homozygous for the nucleic acid encoding a novelpolypeptide.

A suitable group of plants with which to practice the invention includedicots, such as safflower, alfalfa, soybean, rapeseed (high erucic acidand canola), or sunflower. Also suitable are monocots such as corn,wheat, rye, barley, oat, rice, millet, amaranth or sorghum. Alsosuitable are vegetable crops or root crops such as potato, broccoli,peas, sweet corn, popcorn, tomato, beans (including kidney beans, limabeans, dry beans, green beans) and the like. Also suitable are fruitcrops such as peach, pear, apple, cherry, orange, lemon, grapefruit,plum, mango and palm. Thus, the invention has use over a broad range ofplants, including species from the genera Anacardium, Arachis,Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum,Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria,Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus,Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana,Medicago, Nicotiana, Olea, Oryza, Panicum, Pannesetum, Persea,Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale,Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum,Vicia, Vitis, Vigna and Zea.

The nucleic acid molecules of the invention can be expressed in plantsin a cell- or tissue-specific manner according to the regulatoryelements chosen to include in a particular nucleic acid constructpresent in the plant. Suitable cells, tissues, and organs in which toexpress a chimeric polypeptide of the invention include, withoutlimitation, egg cell, central cell, synergid cell, zygote, ovuleprimordia, nucellus, integuments, endothelium, female gametophyte cells,embryo, axis, cotyledons, suspensor, endosperm, seed coat, groundmeristem, vascular bundle, cambium, phloem, cortex, shoot or root apicalmeristems, lateral shoot or root meristems, floral meristem, leafprimordia, leaf mesophyll cells, and leaf epidermal cells, e.g.,epidermal cells involved in forming the cuticular layer. Also suitableare cells and tissues grown in liquid media or on semi-solid media.

Transgenic Fungi

Other eukaryotic organisms featured in the invention are fungicontaining an exogenous nucleic acid molecule that encodes an engineeredRNA precursor of the invention.

Accordingly, a method according to the invention comprises introducing anucleic acid molecule or construct as described herein into a fungus.Techniques for introducing exogenous nucleic acids into many fungi areknown in the art, see, e.g., U.S. Pat. Nos. 5,252,726 and 5,070,020.Transformed fungi can be cultured by techniques known to those skilledin the art. Such fungi can be used to introduce a nucleic acid encodinga polypeptide into other fungal strains, to transfer the nucleic acid toother species or for further selection of other desirable traits.

A suitable group of fungi with which to practice the invention includefission yeast and budding yeast, such as Saccharomyces cereviseae, S.pombe, S. carlsbergeris and Candida albicans. Filamentous fungi such asAspergillus spp. and Penicillium spp. are also useful.

Pharmaceutical Compositions

The molecules of the invention can be incorporated into pharmaceuticalcompositions. Such compositions typically include a nucleic acidmolecule, e.g., a nucleic acid molecule (e.g., a transgene) that encodesan engineered RNA precursor, or the precursor RNA itself, and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” includes solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous,inhalation, transdermal (topical), transmucosal, and rectaladministration. Administration can also be oral. Solutions orsuspensions used for parenteral administration such as intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringes,or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel3, or corn starch; a lubricant such as magnesium stearate orSterotes3; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions of the invention lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

A therapeutically effective amount of a composition containing asequence that encodes an engineered RNA precursor, or the precursoritself (i.e., an effective dosage), is an amount that inhibitsexpression of the polypeptide encoded by the target gene by at least 30percent. Higher percentages of inhibition, e.g., 45, 50, 75, 85, 90percent or higher may be preferred in certain embodiments. Exemplarydoses include milligram or microgram amounts of the molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. The compositions can beadministered one time per week for between about 1 to 10 weeks, e.g.,between 2 to 8 weeks, or between about 3 to 7 weeks, or for about 4, 5,or 6 weeks. The skilled artisan will appreciate that certain factors mayinfluence the dosage and timing required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. In some cases transient expressionof the engineered RNA precursor may be desired. When an induciblepromoter is included in the construct encoding an engineered RNAprecursor, expression is assayed upon delivery to the subject of anappropriate dose of the substance used to induce expression.

It is furthermore understood that appropriate doses of a compositiondepend upon the potency of the molecule (the sequence encoding theengineered precursor) with respect to the expression or activity to bemodulated. When one or more of these molecules is to be administered toan animal (e.g., a human) to modulate expression or activity of apolypeptide or nucleic acid of the invention, a physician, veterinarian,or researcher may, for example, prescribe a relatively low dose atfirst, subsequently increasing the dose until an appropriate response isobtained. In addition, it is understood that the specific dose level forany particular subject will depend upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

The nucleic acid molecules of the invention can be generally insertedinto vectors and used as gene therapy vectors. Gene therapy vectors canbe delivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted expression oractivity of any gene that is transcribed. As used herein, the term“treatment” is defined as the application or administration of atherapeutic agent to a patient, or application or administration of atherapeutic agent to an isolated tissue or cell line from a patient, whohas a disorder, e.g., a disease or condition, a symptom of disease, or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve, or affect thedisease, the symptoms of disease, or the predisposition toward disease.A therapeutic agent is an engineered RNA precursor of the invention, ora nucleic acid molecule (DNA) that encodes the precursor.

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained about a subject's genome, specifically knowledgeabout a gene sequence (i.e., mutated gene) whose expression isassociated with disease. Thus, a molecule of the invention can beengineered, based on knowledge of the gene whose expression is targeted,to inhibit expression of that gene as described herein.

Thus, in one aspect, the invention provides a method for treating in asubject, a disorder, e.g., a disease or condition, associated with anaberrant or unwanted gene expression or activity, by administering tothe subject an engineered nucleic acid sequence that encodes anengineered precursor RNA. Subjects at risk for a disrder which is causedor contributed to by aberrant or unwanted expression or activity of agene can be identified by, for example, any or a combination ofdiagnostic or prognostic assays that are known in the art.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the aberrance, such that adisease or disorder is prevented or, alternatively, delayed in itsprogression.

The molecules of the invention can act as novel therapeutic agents forcontrolling one or more of cellular proliferative and/or differentiativedisorders, disorders associated with bone metabolism, immune disorders,hematopoietic disorders, cardiovascular disorders, liver disorders,viral diseases, pain or metabolic disorders.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver origin.

As used herein, the terms “cancer,” “hyperproliferative,” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair.

The terms “cancer” or “neoplasms” include malignancies of the variousorgan systems, such as affecting lung, breast, thyroid, lymphoid,gastrointestinal, and genito-urinary tract, as well as adenocarcinomaswhich include malignancies such as most colon cancers, renal-cellcarcinoma, prostate cancer and/or testicular tumors, non-small cellcarcinoma of the lung, cancer of the small intestine and cancer of theesophagus.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

The term “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit. Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

In general, the engineered RNA precursors of the invention are designedto target genes associated with particular disorders. Examples of suchgenes associated with proliferative disorders that can be targetedinclude activated ras, p53, BRCA-1, and BRCA-2. Other specific genesthat can be targeted are those associated with amyotrophic lateralsclerosis (ALS; e.g., superoxide dismutase-1 (SOD1)); Huntington'sdisease (e.g., huntingtin), Parkinson's disease (parkin), and genesassociated with autosomal dominant disorders.

The engineered RNAs of the invention can be used to treat a variety ofimmune disorders, in particular those associated with overexpression ofa gene or expression of a mutant gene. Examples of hematopoieiticdisorders or diseases include, but are not limited to, autoimmunediseases (including, for example, diabetes mellitus, arthritis(including rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjögren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerativecolitis, asthma, allergic asthma, cutaneous lupus erythematosus,scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversalreactions, erythema nodosum leprosum, autoimmune uveitis, allergicencephalomyelitis, acute necrotizing hemorrhagic encephalopathy,idiopathic bilateral progressive sensorineural hearing loss, aplasticanemia, pure red cell anemia, idiopathic thrombocytopenia,polychondritis, Wegener's granulomatosis, chronic active hepatitis,Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, andinterstitial lung fibrosis), graft-versus-host disease, cases oftransplantation, and allergy such as, atopic allergy.

Examples of disorders involving the heart or “cardiovascular disorder”include, but are not limited to, a disease, disorder, or state involvingthe cardiovascular system, e.g., the heart, the blood vessels, and/orthe blood. A cardiovascular disorder can be caused by an imbalance inarterial pressure, a malfunction of the heart, or an occlusion of ablood vessel, e.g., by a thrombus. Examples of such disorders includehypertension, atherosclerosis, coronary artery spasm, congestive heartfailure, coronary artery disease, valvular disease, arrhythmias, andcardiomyopathies.

Disorders which may be treated by methods described herein include, butare not limited to, disorders associated with an accumulation in theliver of fibrous tissue, such as that resulting from an imbalancebetween production and degradation of the extracellular matrixaccompanied by the collapse and condensation of preexisting fibers.

Additionally, molecules of the invention can be used to treat viraldiseases, including but not limited to hepatitis B, hepatitis C, herpessimplex virus (HSV), HIV-AIDS, poliovirus, and smallpox virus. Moleculesof the invention are engineered as described herein to target expressedsequences of a virus, thus ameliorating viral activity and replication.The molecules can be used in the treatment and/or diagnosis of viralinfected tissue. Also, such molecules can be used in the treatment ofvirus-associated carcinoma, such as hepatocellular cancer.

Uses of Engineered RNA Precursors to Induce RNAi

Engineered RNA precursors, introduced into cells or whole organisms asdescribed herein, will lead to the production of a desired siRNAmolecule. Such an siRNA molecule will then associate with endogenousprotein components of the RNAi pathway to bind to and target a specificmRNA sequence for cleavage and destruction. In this fashion, the mRNA tobe targeted by the siRNA generated from the engineered RNA precursorwill be depleted from the cell or organism, leading to a decrease in theconcentration of the protein encoded by that mRNA in the cell ororganism.

For example, one may be seeking to discover a small molecule thatreduces the activity of a kinase whose overexpression leads tounrestrained cell proliferation. This kinase is overexpressed in avariety of cancer cells. A key question to be determined is whether ornot decreasing the activity of this kinase in adult mammals would haveunexpected deleterious effects. By expressing an engineered RNAprecursor that targets for destruction by the RNAi pathway the mRNAencoding the kinase throughout the tissues of an adult mouse, thedeleterious effects of such a potential drug can be determined. That is,the method described here will allow rapid assessment of the suitabilityof the kinase as a drug target.

The new nucleic acid molecules that encode the engineered RNA precursorscan also be used to create large numbers of cells or vectors inmicroarrays in which each cell or vector in the array includes nucleicacid molecules that encode an engineered RNA precursor that is specificfor a different target gene. See, e.g., Ziauddin et al., Nature,411:107-110 (2001).

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Producing an Engineered RNA Precursor

To produce an engineered RNA precursor that will target a gene forfirefly luciferase for cleavage, the sequence of the coding portion ofthe mRNA from firefly luciferase was examined to select a suitablesequence. At a position more than 100 nucleotides, but less than 300nucleotides, 3′ to the start of translation, the sequenceCGUACGCGGAAUACU-UCGAUU (SEQ ID NO:16) was found immediately after thesequence AA. Since this sequence meets the criteria for selection of ansiRNA, it was chosen. An engineered precursor RNA was then designed thatincludes this sequence (underlined) as represented below (and shown inFIG. 2B):

(SEQ ID NO: 2) 5′-GGCAAACGUACGCGGAAUACUUCGAUUAGUAAUUACACAUCAUAA-UCGAAG UAUUCCGCGUACGUUUGCU-3′

Synthetic deoxyoligonucleotide sequences were prepared that serve as anin vitro transcription template for preparing this engineered RNAprecursor using the enzyme T7 RNA Polymerase according to publishedprotocols. Below are shown the two deoxyoligonucleotides. Theoligonucleotides contain the sequence of the T7 RNA Polymerase promoter(underlined in the top strand) to facilitate in vitro transcription intoRNA.

Top oligo: (SEQ ID NO: 17)5′-GCGTAATACGACTCACTATAGGCAAACGTACGCGGAATACTTCGAT-TAGTAATTACACATCATAATCGAAGTATTCCGCGTACGTTTGCT-3′ Bottom oligo:(SEQ ID NO: 18) 5′-TGTAGTCACGTACGCGGAATACTTCGAAGAAACGAGTAATTACTAA-ATCGAAGTATTCCGCGTACGTTTGCCTATAGTGAGTCGTATTACGC-3′

Next, the double-stranded DNA was formed by annealing the twodeoxyoligonucleotides and was transcribed into RNA using T7 RNAPolymerase. The resulting engineered RNA precursor was purified bystandard means, and then tested for its ability to promote cleavage ofthe target mRNA in vitro in a standard RNAi reaction.

Briefly, firefly luciferase mRNA was prepared by in vitro transcriptionand radiolabeled using α-³²P-GTP and Guanylyl Transferase as describedpreviously (Tuschl et al., (1999) cited supra) to generate a radioactivetarget mRNA. The radioactive target mRNA was incubated in a standard invitro RNAi reaction with Drosophila embryo lysate and 50 nM engineeredRNA precursor (ESP) for 0 and 3 hours as described previously (Tuschl etal., (1999) cited supra). The reaction products were isolated andanalyzed by denaturing acrylamide gel electrophoresis as describedpreviously (Zamore et al., (2000) cited supra). As shown in FIG. 3, theengineered RNA precursor induced sequence-specific cleavage of theradioactive target mRNA (5′ cleavage product). Thus, the precursor wasshown to mediate RNAi. FIG. 3 also shows the RNA cleavage product of astandard siRNA (also incubated for 0 and 3 hours), which produced thesame sequence-specific 5′ cleavage product as the ESP.

Example 2 Engineered Let-7 Precursor RNAs Asymmetrically Trigger RNAi InVitro

Two engineered let-7 RNA precursors (ESPs) were prepared in which thestem of pre-let-7 (FIGS. 4B and 4C) was altered to contain a sequencecomplementary to firefly luciferase. Because most stRNAs begin withuracil, the ESPs were designed so that the luciferase-complementarysequence (anti-sense luciferase) began with U. Since stRNAs can beencoded on either the 5′ or the 3′ side of the precursor hairpin (e.g.,on either stem) the anti-sense luciferase sequence (in bold) was placedon the 3′ side of the stem in one ESP (3′ ESP) (FIG. 4B) and on the 5′side in the second stem (5′ ESP) (FIG. 4C).

The ESP RNAs were prepared as generally described in Example 1 by usingthe following DNA oligonucleotide pairs to generate partiallysingle-stranded T7 RNA Polymerase transcription templates:

(SEQ ID NO: 19) 5′-GTAATACGACTCACTATAG-3′ (SEQ ID NO: 20)5′-GGCAAATTCGAAGTATTCCGCGTACGTGATGATGTGTAATTACTCACGTACGCGGAATACTTCGAATTTGCCTATAGTGAGTCGTATTAC-3′ (5′ ESP) (SEQ ID NO: 21)5′-GGCAAATCGTACGCGGAATACTTCGAAAATGATGTGTAATTACTTTTCGAAGTATTCCGCGTACGATTTGCCTATAGTGAGTCGTATTAC-3′ (3′ ESP)

The anti-sense firefly luciferase target RNA has been describedpreviously (A. Nykänen, B. Haley, P. D. Zamore, Cell 107, 309, 2001).

The ability of each ESP to direct luciferase-specific RNAi in an invitro reaction was tested against a target mRNA (shown schematically inFIG. 4D) containing a portion of the firefly luciferase mRNA and asequence fully complementary to let-7 (the target was constructed bystandard techniques and synthesized using T7 RNA Polymerase). As acontrol, an siRNA duplex containing the anti-sense luciferase sequencewas used (FIG. 4A).

FIG. 4E is an autoradiograph showing the results of an assay fordetermining whether the 5′ and 3′ ESPs can equally promote cleavage ofthe target RNA in vitro (the assay conditions are described in Example1, except the ESPs and control were incubated for 0 and 2 hours). Boththe 3′ and the 5′ ESPs directed cleavage of the target RNA within theluciferase sequences, the same site cleaved when the RNAi reaction wasprogrammed with the control siRNA.

Example 3 Preparing a Transgene Encoding an Engineered RNA Precursor

To prepare a transgene encoding an engineered precursor to targetdestruction of the luciferase mRNA in a transgenic mouse that expressesfirefly luciferase mRNA in all of its cells, the engineered RNAprecursor sequence described in Example 1 is cloned by standardrecombinant DNA methods into a nucleic acid molecule, e.g., a vectorcontaining a constitutively expressed promoter sequence and the desirednucleic acid sequence (transgene) encoding the engineered RNA precursoras illustrated in FIG. 5. This vector will also contain sequencesappropriate for its introduction into ES cells to produce transgenicmice by standard methods. The resulting transgene expresses theengineered RNA precursor in all cells of a transgenic mouse.

The engineered precursor RNA is then processed by Dicer and othercomponents of the RNAi machinery to yield an siRNA directed against thefirefly luciferase gene. This siRNA directs cleavage of the luciferasemRNA, resulting in a decrease in the expression of luciferase mRNA inthe cells of the animal.

The same methods can be used to silence other target genes, either usingconstitutively expressed or inducible expression systems in a variety oftransgenic animals.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. An adeno-associated viral (AAV) vector comprisingan isolated nucleic acid molecule comprising a regulatory sequenceoperably linked to a nucleic acid sequence that encodes an engineeredribonucleic acid (RNA) precursor which is processed to form a smallinterfering ribonucleic acid (siRNA) comprising an antisense strandcomplementary to a sequence of a messenger RNA (mRNA) of a target geneand a sense strand having a sequence complementary to the antisensestrand, wherein the precursor comprises (i) a first stem portionconsisting of a sequence of about 18 to about 40 nucleotides that iscomplementary to a sequence of the of the mRNA of the target gene; (ii)a second stem portion consisting of a sequence of about 18 to about 40nucleotides that is sufficiently complementary to the about 18 to about40 nucleotide sequence of the first stem portion to hybridize with thefirst stem portion to form a duplex stem; and (iii) a loop portionconsisting of 2 to 9 nucleotides that connects the two stem portions. 2.The AAV vector of claim 1, wherein the second stem portion is fullycomplementary to the first stem portion.
 3. The AAV vector of claim 1,wherein the first stem portion is located at the 5′ end of the RNAprecursor or wherein the first stem portion is located at the 3′ end ofthe RNA precursor.
 4. The AAV vector of claim 1, wherein the loopportion consists of at least 4 nucleotides.
 5. The AAV vector of claim4, wherein the first and second stem portions each consist of 18, 19,20, 21 or 22 nucleotides.
 6. The AAV vector of claim 1, wherein thesequence of the mRNA to which the sequence of about 18 to about 40nucleotides is complementary, is located from 100 to 300 nucleotides 3′of the start of translation of the mRNA of the target gene or whereinthe sequence of the mRNA to which the sequence of about 18 to about 40nucleotides is complementary, is located in a 5′ untranslated region(UTR) or a 3′ UTR of the mRNA of the target gene.
 7. The AAV vector ofclaim 1, wherein the first and second stem portions each consist ofabout 18 to about 30 nucleotides.
 8. The AAV vector of claim 1, whereinthe first and second stem portions each comprise the same number ofnucleotides or wherein one of the first and second stem portionscomprises 1 to 4 more nucleotides than the other stem portion.
 9. TheAAV vector of claim 1, wherein the regulatory sequence comprises a PolIII or Pol II promoter.
 10. The AAV vector of claim 1, wherein theregulatory sequence is constitutive or inducible.
 11. The AAV vector ofclaim 1, wherein the loop portion consists of at least 4 nucleotides orconsists of at least 7 nucleotides.
 12. The AAV vector of claim 1,wherein the first and second stem portions each consist of 22 to about28 nucleotides.
 13. The AAV vector of claim 1, wherein the first stemportion consists essentially of the antisense strand of the siRNAflanked by sequences not having complementarity to the sequence of themRNA, wherein the second stem portion consists essentially of the sensestrand of the siRNA flanked by sequences not sharing identity with thesequence of the mRNA, and wherein the sequences flanking the antisenseand sense strands in the stem portions facilitate processing of theprecursor in a mammalian cell to form the siRNA.
 14. A host cellcomprising the AAV vector of claim
 1. 15. A method of inducingribonucleic acid interference (RNAi) of a target gene in a mammaliancell in vivo, the method comprising (a) contacting the cell with the AAVvector of claim 1; and (b) inducing the cell to express the engineeredRNA precursor encoded by the nucleic acid molecule; wherein theprecursor is processed by the cell to generate the siRNA that mediatescleavage of the mRNA, thereby inducing RNAi of the target gene in themammalian cell in vivo.
 16. The method of claim 15, wherein the targetgene is a human gene.
 17. The method of claim 15, wherein the targetgene is a mutant human gene or a viral gene.
 18. An engineeredribonucleic acid (RNA) precursor which is processed to form a smallinterfering ribonucleic acid (siRNA) comprising an antisense strandcomplementary to a sequence of a messenger RNA (mRNA) of a target geneand a sense strand having a sequence complementary to the antisensestrand, said engineered RNA precursor comprising, (i) a first stemportion consisting of a sequence of about 18 to about 40 nucleotidesthat is complementary to a sequence of the mRNA of the target gene; (ii)a second stem portion consisting of a sequence of about 18 to about 40nucleotides that is sufficiently complementary to the about 18 to about40 nucleotide sequence of the first stem portion to hybridize with thefirst stem portion to form a duplex stem; and (iii) a loop portionconsisting of 2 to 9 nucleotides that connects the two stem portions.19. The engineered RNA precursor of claim 18, wherein the second stemportion is fully complementary to the first stem portion.
 20. Theengineered RNA precursor of claim 18, wherein the first stem portion islocated at the 5′ end of the RNA precursor or wherein the first stemportion is located at the 3′ end of the RNA precursor.
 21. Theengineered RNA precursor of claim 18, wherein the loop portion consistsof at least 4 nucleotides.
 22. The engineered RNA precursor of claim 21,wherein the first and second stem portions each consist of 18, 19, 20,21 or 22 nucleotides.
 23. The engineered RNA precursor of claim 18,wherein the sequence of the mRNA to which the sequence of about 18 toabout 40 nucleotides is complementary, is located from 100 to 300nucleotides 3′ of the start of translation of the mRNA of the targetgene or wherein the sequence of the mRNA to which the sequence of about18 to about 40 nucleotides is complementary, is located in a 5′untranslated region (UTR) or a 3′ UTR of the mRNA of the target gene.24. The engineered RNA precursor of claim 18, wherein the first andsecond stem portions each consist of about 18 to about 30 nucleotides.25. The engineered RNA precursor of claim 18, wherein the first andsecond stem portions each comprise the same number of nucleotides orwherein one of the first and second stem portions comprises 1 to 4 morenucleotides than the other stem portion.
 26. The engineered RNAprecursor of claim 18, wherein the loop portion consists of at least 4nucleotides or consists of at least 7 nucleotides.
 27. The engineeredRNA precursor of claim 18, wherein the first and second stem portionseach consist of 22 to about 28 nucleotides.
 28. The engineered RNAprecursor of claim 18, wherein the first stem portion consistsessentially of the antisense strand of the siRNA flanked by sequencesnot having complementarity to the sequence of the mRNA, wherein thesecond stem portion consists essentially of the sense strand of thesiRNA flanked by sequences not sharing identity with the sequence of themRNA, and wherein the sequences flanking the antisense and sense strandsin the stem portions facilitate processing of the precursor in amammalian cell to form the siRNA.
 29. A method of inducing ribonucleicacid interference (RNAi) of a target gene in a mammalian cell in vitro,the method comprising 18 (a) contacting the cell with the engineered RNAprecursor of claim 18; and (b) maintaining the cell under conditionssufficient to permit processing of the precursor by the cell to generatethe siRNA that mediates cleavage of the mRNA, thereby inducing RNAi ofthe target gene in the mammalian cell in vitro.
 30. The method of claim29, wherein the target gene is a human gene.
 31. The method of claim 29,wherein the target gene is a mutant human gene or a viral gene.
 32. Anisolated nucleic acid molecule comprising a regulatory sequence operablylinked to a nucleic acid sequence that encodes an engineered ribonucleicacid (RNA) precursor which is processed to form a small interferingribonucleic acid (siRNA) comprising an antisense strand complementary toa sequence of a messenger RNA (mRNA) of a target gene and a sense strandhaving a sequence complementary to the antisense strand, wherein theprecursor comprises (i) a first stem portion consisting of a sequence ofabout 18 to about 40 nucleotides that is complementary to a sequence ofthe of the mRNA of the target gene; (ii) a second stem portionconsisting of a sequence of about 18 to about 40 nucleotides that issufficiently complementary to the about 18 to about 40 nucleotidesequence of the first stem portion to hybridize with the first stemportion to form a duplex stem; and (iii) a loop portion that connectsthe two stem portions, wherein the loop portion consists of 2 to 9nucleotides.
 33. The method of claim 32, wherein the target gene is ahuman gene.
 34. The method of claim 32, wherein the target gene is amutant human gene or a viral gene.
 35. The isolated nucleic acidmolecule of claim 32, wherein the first stem portion consistsessentially of the antisense strand of the siRNA flanked by sequencesnot having complementarity to the sequence of the mRNA, wherein thesecond stem portion consists essentially of the sense strand of thesiRNA flanked by sequences not sharing identity with the sequence of themRNA, and wherein the sequences flanking the antisense and sense strandsin the stem portions facilitate processing of the precursor in amammalian cell to form the siRNA.